灰葡萄孢菌过氧化物酶体及细胞核的荧光蛋白标记

Fluorescent labeling of peroxisome and nucleus in Botrytis cinerea

摘要:

[目的] 对灰葡萄孢菌（Botrytis cinerea）的细胞核和过氧化物酶体进行荧光蛋白标记，为研究其生长发育和侵染过程中细胞结构和细胞器动态提供基础。[方法] 以绿色荧光蛋白（GFP）和红色荧光蛋白（DsRED、mCherry）为报告基因，利用根癌农杆菌介导转化（Agrobacterium tumefaciens mediated transformation，AtMT）将3种荧光蛋白标记载体分别导入灰葡萄孢菌标准菌株B05.10；通过PCR检测及荧光观察筛选和验证转化子，并进行单孢纯化；利用共聚焦显微镜记录细胞器荧光定位情况。[结果] 获得了过氧化物酶体或细胞核稳定表达红、绿色荧光的重组单孢菌株，PCR验证表明标记基因成功整合入转化子基因组。在标记细胞核的菌株中，菌丝和孢子中可见多个明亮、圆形的荧光点，与DAPI染色共定位。标记过氧化物酶体的菌株中，菌丝和孢子中可见小点状绿色或红色荧光，在脂类物质诱导下荧光点数量明显增加，符合过氧化物酶体分布及动态特征。细胞壁染色结果显示，细胞壁染色产生的蓝色荧光与红、绿荧光蛋白的荧光互不干扰，标记效果良好。[结论] 获得了理想的过氧化物酶体或细胞核荧光标记的灰葡萄孢菌菌株，为研究其细胞器动态以及生长发育与致病分子机制提供了参考和材料。

关键词:灰葡萄孢菌;荧光蛋白标记;细胞核;过氧化物酶体

Abstract:

[Objective] To label the nuclei and peroxisomes of Botrytis cinerea with fluorescent proteins, as a tool for further investigation on biogenesis and dynamics of the cellular structures of the fungus. [Methods] The green fluorescent protein (GFP) and the red fluorescent proteins (DsRed and mCherry) were used as reporter proteins to label the nuclei and peroxisomes in B. cinerea. Three fluorescent labeling vectors were separately introduced into the B. cinerea strain B05.10 via Agrobacterium tumefaciens-mediated transformation. The resulting transformants were selected and confirmed by PCR, and then analyzed with the confocal fluorescent microscope. [Results] The single-spore purified recombinant strains expressing red or green fluorescence were obtained. The PCR amplification and fluorescence detection indicated that the fluorescent genes were integrated into the genome of the transformants. Round fluorescent spots were detected in mycelia and conidia of the strains with nuclear labeling, overlapping well with DAPI staining. In the strains with peroxisomes labeling, small green or red fluorescent dots were present in mycelia and conidia. Upon induction on lipids, the number of the dots was significantly increased, corresponding well with the described distribution profile of peroxisomes. In addition, the blue fluorescence produced by Calcofluor white staining did not interfere with the fluorescence of red or green fluorescent proteins, capable of forming well-integrated multi-fluorescent images. [Conclusion] We obtained the B. cinerea strains with fluorescent labeled nuclei or peroxisomes, which are maybe ideal tools for studying the biogenesis and dynamics of cellular organelles, the developments and even the pathogenesis of the fungus.

Key words:Botrytis cinerea;fluorescent protein labelling;nucleus;peroxisome